Susan Zolla-Pazner
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Researcher at Icahn School of Medicine at Mount Sinai
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SARS-CoV-2, commonly termed COVID-19 for the illness it causes, has infected >3.2 million people, including >220,000 deaths. Human milk IgG originates mainly from blood, therefore a SARS-CoV-2-reactive antibody (Ab) response in milk would be expected (1). However, IgG comprises only ~2% of milk Ab, with most milk Abs originating from mucosa-associated lymphatic tissue (1). Therefore, the extent of the milk immune response to SARS-CoV-2 is unknown (2). This response is critical for infants and young children, who tend not to suffer greatly from COVID-19 pathology but are likely responsible for significant virus transmission (3-5). Perhaps even more significant is the fact that milk Abs could be purified and used as a COVID-19 therapeutic, given they would likely be of the secretory (s) class and highly resistant to proteolytic degradation in the respiratory tissue (2, 6). In this preliminary report, 15 milk samples obtained from donors previously-infected with SARS-CoV-2 as well as 10 negative control samples obtained prior to December 2019 were tested for reactivity to the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein by ELISAs measuring IgA, IgG, IgM, and secretory Ab. Eighty percent of samples obtained post-COVID-19 exhibited IgA reactivity, and all these samples were also positive for secretory Ab reactivity, suggesting the IgA is predominantly sIgA. COVID-19 group mean OD values of undiluted milk were significantly greater for IgA (p<0.0001), secretory-type Abs (p<0.0001), and IgG (p=0.017), but not for IgM, compared to pre-pandemic group mean values. Overall, these data indicate that there is strong sIgA-dominant SARS-CoV-2 immune response in human milk after infection in the majority of individuals, and that a comprehensive study of this response is highly warranted.
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Approximately 10% of infants will experience COVID19 illness requiring advanced care (1). A potential mechanism to protect this population could be provided by passive immunity through the milk of a previously infected mother. We and others have reported on the presence of SARS-CoV-2-specific antibodies in human milk (2-5). We now report the prevalence of SARS-CoV-2 IgA in the milk of 75 COVID-19-recovered participants, and find that 88% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high-titer. These IgA positive specimens were also positive for Spike-specific antibodies bearing the secretory component. Levels of IgA antibodies and antibodies bearing secretory component were shown to be strongly positively correlated. The secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases. Our IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC50 range, 2.39-89.4ug/mL) compared to 1 of 8 controls. IgA binding and neutralization capacities were found to be strongly positively correlated. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly stable not only in milk and the infant mouth and gut, but in all mucosa including the gastrointestinal tract, upper airway, and lungs (6).
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The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an out-of-the-box VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.
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Background. More than one million infections with the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) have been confirmed. While PCR-based assays are used for diagnosis, high through-put serologic methods are needed to detect antibodies for seroserveillance and for identification of seroconversion, potential plasma donors, and the nature of the immune response to this pathogen. Methods. A Luminex binding assay was used to assess the presence of antibodies in human sera from COVID-19-infected and -uninfected individuals specific for two recombinant proteins of SARS-CoV-2. Findings. Fluorochrome-labeled beads were coated with a recombinant soluble stabilized trimeric SARS-CoV-2 S protein ectodomain or its central portion, the receptor binding domain (RBD). Coated beads were incubated with sera, followed by incubation with biotinylated anti-human total Ig antibodies and phycoerythrin (PE)-labeled streptavidin. Readout using a Luminex analyzer clearly differentiated between sera of the infected and uninfected subject, delineating a wide range of serum antibody levels in infected subjects. Interpretation. Antibody assays of sera can identify individuals who are infected with SARS-CoV-2 and have seroconverted, as well as subjects who have been infected and recovered. The use of the Luminex binding Ab assay has the advantage that it can be run in approximately 2.5 hours, uses very little antigen, and permits a high through-put of samples/day. Funding. NIAID contracts and grants, Department of Veterans Affairs grants, the Microbiology Laboratory Clinical Services, Translational Science Hub, and Personalized Virology Initiative, and Department of Medicine of Mount Sinai Health System and Icahn School of Medicine at Mount Sinai.